Ca²⁺-induced enzyme and fluid secretion in pancreatic acini is not completely understood and may involve activation of ion permeabilities in zymogen granule (ZG) membranes. We hypothesized that a chromanol 293B-sensitive K⁺ conductance carried by KCNQ1 is expressed in ZG membranes (ZGM). In rat pancreatic ZG suspended in isotonic salts ion flux was determined by the rate of ionophore-induced osmotic lysis. The KCNQ1 inhibitor 293B selectively blocked K⁺ conductance (IC₅₀ ~10µM). Upon incorporation of ZG membranes into planar bilayers, cation channels were detected in 645/150mM K-gluconate cis/trans solutions. Channels had linear current-voltage relationships, a reversal potential (E_rev) of -20.9±0.9 mV and a single channel conductance (g_K) of 265.8±44.0 pS (n=39). Replacement of cis 500mM K⁺ by 500mM Na⁺ shifted E_rev to -2.4±3.6 mV (n=3), indicating K⁺ selectivity. Single channel analysis identified several K⁺ channel groups with distinct channel behaviors. K⁺ channels with a g_K of 651.8±88.0 pS, E_rev of -22.9±2.2 mV and open probability (P_open) of 0.43±0.06 at 0 mV (n=6) as well as channels with a g_K of 155.0±11.4 pS, E_rev of -18.3±1.8 mV and P_open of 0.80±0.03 at 0 mV (n=3) were inhibited by 100µM 293B or by the selective KCNQ1 inhibitor HMR1556, but not by the maxi K_Ca channel inhibitor charybdotoxin. KCNQ1 was demonstrated by immunoperoxidase labelling of pancreatic tissue, immunogold labelling of ZG and immunoblotting of ZG membranes. 293B also inhibited cholecystokinin-induced amylase secretion of permeabilized acini (IC₅₀ ~10µM). KCNQ1 may account for ZG K⁺ conductance and contribute to hormone-stimulated enzyme and fluid secretion by the pancreas.